SRM/MRM assay for the fatty acid synthase protein

ABSTRACT

Specific peptides, and derived ionization characteristics of the peptides, from the Fatty acid synthase (FASN) protein are provided that are particularly advantageous for quantifying the FASN protein directly in biological samples that have been fixed in formalin by the method of Selected Reaction Monitoring (SRM) mass spectrometry, or what can also be termed as Multiple Reaction Monitoring (MRM) mass spectrometry. Such biological samples are chemically preserved and fixed and are selected from tissues and cells treated with formaldehyde containing agents/fixatives including formalin-fixed tissue/cells, formalin-fixed/paraffin embedded (FFPE) tissue/cells, FFPE tissue blocks and cells from those blocks, and tissue culture cells that have been formalin fixed and or paraffin embedded. A protein sample is prepared from said biological sample using the Liquid Tissue™ reagents and protocol and the FASN protein is quantitated in the Liquid Tissue™ sample by the method of SRM/MRM mass spectrometry by quantitating in the protein sample at least one or more of the peptides described. These peptides can be quantitated if they reside in a modified or an unmodified form. An example of a modified form of an FASN peptide is phosphorylation of a tyrosine, threonine, serine, and/or other amino acid residues within the peptide sequence.

This application is a continuation of International Application No.PCT/US12/56959, filed Sep. 24, 2012, which claims the benefit of U.S.Provisional Application No. 61/538,091, filed Sep. 22, 2011, entitled“SRM/MRM Assay for the Fatty Acid Synthase Protein,” the contents ofeach of which are hereby incorporated by reference in their entireties.This application also contains a sequence listing submittedelectronically via EFS-web, which serves as both the paper copy and thecomputer readable form (CRF) and consists of a file entitled “0011528024 US01 SEQ LISTING”, which was created on Mar. 19, 2014, which is215,040 bytes in size, and which is also incorporated by reference inits entirety.

INTRODUCTION

Specific peptides derived from subsequences of the Fatty Acid Synthaseprotein (referred to herein as FASN, and which also is referred to asFAS), are provided. The peptide sequence and fragmentation/transitionions for each peptide are particularly useful in a massspectrometry-based Selected Reaction Monitoring (SRM) assay, which canalso be referred to as a Multiple Reaction Monitoring (MRM) assay. Suchassays are referred to herein as SRM/MRM. The use of peptides forquantitative SRM/MRM analysis of the FASN protein is described.

This SRM/MRM assay can be used to measure relative or absolutequantitative levels of one or more of the specific peptides from theFASN protein. This provides a means of measuring the amount of the FASNprotein in a given protein preparation obtained from a biological sampleby mass spectrometry.

More specifically, the SRM/MRM assay can measure these peptides directlyin complex protein lysate samples prepared from cells procured frompatient tissue samples, such as formalin fixed cancer patient tissue.Methods of preparing protein samples from formalin-fixed tissue aredescribed in U.S. Pat. No. 7,473,532, the contents of which are herebyincorporated by reference in their entirety. The methods described inU.S. Pat. No. 7,473,532 may conveniently be carried out using LiquidTissue™ reagents and protocol available from OncoPlexDx (formerlyExpression Pathology Inc., Rockville, Md.).

The most widely and advantageously available form of tissues from cancerpatients tissue is formalin fixed, paraffin embedded tissue.Formaldehyde/formalin fixation of surgically removed tissue is by farand away the most common method of preserving cancer tissue samplesworldwide and is the accepted convention for standard pathologypractice. Aqueous solutions of formaldehyde are referred to as formalin.“100%” formalin consists of a saturated solution of formaldehyde (thisis about 40% by volume or 37% by mass) in water, with a small amount ofstabilizer, usually methanol to limit oxidation and degree ofpolymerization. The most common way in which tissue is preserved is tosoak whole tissue for extended periods of time (8 hours to 48 hours) inaqueous formaldehyde, commonly termed 10% neutral buffered formalin,followed by embedding the fixed whole tissue in paraffin wax for longterm storage at room temperature. Thus, molecular analytical methods toanalyze formalin fixed cancer tissue will be the most accepted andheavily utilized methods for analysis of cancer patient tissue.

Results from the SRM/MRM assay can be used to correlate accurate andprecise quantitative levels of the FASN protein within the specifictissue samples (e.g., one or more cancer tissue samples) of the patientor subject from whom the tissue (biological sample) was collected andpreserved. This not only provides diagnostic information about thecancer, but also permits a physician or other medical professional todetermine appropriate therapy for the patient. For example, such anassay can be designed to diagnose the stage or degree of a cancer anddetermine a therapeutic agent to which a patient is most likely torespond. Such an assay that provides diagnostically and therapeuticallyimportant information about levels of protein expression in a diseasedtissue or other patient sample is termed a companion diagnostic assay.

SUMMARY

The assays described herein measure relative or absolute levels ofspecific unmodified peptides from the FASN protein and also can measureabsolute or relative levels of specific modified peptides from the FASNprotein. Examples of modifications include phosphorylated amino acidresidues (e.g. phosphotyrosine, phosphoserine and phosphothreonine) andglycosylated amino acid residues (e.g. glycosylated asparagine residues)that are present on the peptides.

Relative quantitative levels of the FASN protein are determined by theSRM/MRM methodology, for example, by comparing SRM/MRM signature peakareas (e.g., signature peak area or integrated fragment ion intensity)of an individual FASN peptide in different samples. Alternatively, it ispossible to compare multiple SRM/MRM signature peak areas for multipleFASN signature peptides, where each peptide has its own specific SRM/MRMsignature peak, to determine the relative FASN protein content in onebiological sample with the FASN protein content in one or moreadditional or different biological samples. In this way, the amount of aparticular peptide, or peptides, from the FASN protein, and thereforethe amount of the FASN protein, is determined relative to the same FASNpeptide, or peptides, across 2 or more biological samples under the sameexperimental conditions. In addition, relative quantitation can bedetermined for a given peptide, or peptides, from the FASN proteinwithin a single sample by comparing the signature peak area for thatpeptide by SRM/MRM methodology to the signature peak area for anotherand different peptide, or peptides, from a different protein, orproteins, within the same protein preparation from the biologicalsample. In this way, the amount of a particular peptide from the FASNprotein, and therefore the amount of the FASN protein, is determinedrelative one to another within the same sample. These approachesgenerate quantitation of an individual peptide, or peptides, from theFASN protein to the amount of another peptide, or peptides, betweensamples and within samples wherein the amounts as determined by peakarea are relative one to another, regardless of the absolute weight tovolume or weight to weight amounts of the FASN peptide in the proteinpreparation from the biological sample. Relative quantitative data aboutindividual signature peak areas between different samples are normalizedto the amount of protein analyzed per sample. Relative quantitation canbe performed across many peptides from multiple proteins and the FASNprotein simultaneously in a single sample and/or across many samples togain insight into relative protein amounts, one peptide/protein withrespect to other peptides/proteins.

Absolute quantitative levels of the FASN protein are determined by, forexample, the SRM/MRM methodology whereby the SRM/MRM signature peak areaof an individual peptide from the FASN protein in one biological sampleis compared to the SRM/MRM signature peak area of an exogenously added“spiked” internal standard. In one embodiment, the internal standard isa synthetic version of the same exact FASN peptide that contains one ormore amino acid residues labeled with one or more heavy isotopes.Suitable isotope-labeled internal standards are synthesized so that,when analyzed by mass spectrometry, each standard generates apredictable and consistent SRM/MRM signature peak that is different anddistinct from the native FASN peptide signature peak and which can beused as a comparator peak. Thus, when the internal standard is spiked ina known amount into a protein preparation from a biological sample andanalyzed by mass spectrometry, the SRM/MRM signature peak area of thenative peptide from the sample can be compared to the SRM/MRM signaturepeak area of the internal standard peptide. This numerical comparisonprovides either the absolute molarity and/or absolute weight of thenative peptide present in the original protein preparation from thebiological sample. Absolute quantitative data for fragment peptides aredisplayed according to the amount of protein analyzed per sample.Absolute quantitation can be performed across many peptides, and thusproteins, simultaneously in a single sample and/or across many samplesto gain insight into absolute protein amounts in individual biologicalsamples and in entire cohorts of individual samples.

The SRM/MRM assay method can be used to aid diagnosis of the stage ofcancer, for example, directly in patient-derived tissue, such asformalin fixed tissue, and to aid in determining which therapeutic agentwould be most advantageous for use in treating that patient. Cancertissue that is removed from a patient either through surgery, such asfor therapeutic removal of partial or entire tumors, or through biopsyprocedures conducted to determine the presence or absence of suspecteddisease, is analyzed to determine whether or not a specific protein, orproteins, and which forms of proteins, are present in that patienttissue. Moreover, the expression level of a protein, or multipleproteins, can be determined and compared to a “normal” or referencelevel found in healthy tissue. Normal or reference levels of proteinsfound in healthy tissue may be derived from, for example, the relevanttissues of one or more individuals that do not have cancer.Alternatively, normal or reference levels may be obtained forindividuals with cancer by analysis of relevant tissues not affected bythe cancer.

Assays of protein levels (e.g., FASN levels) can also be used todiagnose the stage of cancer in a patient or subject diagnosed withcancer by employing the FASN levels. Levels or amounts of proteins orpeptides can be defined as the quantity expressed in moles, mass orweight of a protein or peptide determined by the SRM/MRM assay. Thelevel or amount may be normalized to total the level or amount ofprotein or another component in the lysate analyzed (e.g., expressed inmicromoles/microgram of protein or micrograms/microgram of protein). Inaddition, the level or amount of a protein or peptide may be determinedon volume basis, expressed, for example, in micromolar ornanograms/microliter. The level or amount of protein or peptide asdetermined by the SRM/MRM assay can also be normalized to the number ofcells analyzed. Information regarding FASN can thus be used to aid indetermining stage or grade of a cancer by correlating the level of theFASN protein (or fragment peptides of the FASN protein) with levelsobserved in normal tissues.

Once the stage and/or grade, and/or FASN protein expressioncharacteristics of the cancer has been determined, that information canbe matched to a list of therapeutic agents (chemical and biological)developed to specifically treat cancer tissue that is characterized by,for example, abnormal expression of the protein or protein(s) (e.g.,FASN) that were assayed. Matching information from an FASN protein assayto a list of therapeutic agents that specifically targets, for example,the FASN protein or cells/tissue expressing the protein, defines whathas been termed a personalized medicine approach to treating disease.The assay methods described herein form the foundation of a personalizedmedicine approach by using analysis of proteins from the patient's owntissue as a source for diagnostic and treatment decisions.

DETAILED DESCRIPTION

In principle, any predicted peptide derived from FASN protein, preparedfor example by digesting with a protease of known specificity (e.g.trypsin), can be used as a surrogate reporter to determine the abundanceof FASN protein in a sample using a mass spectrometry-based SRM/MRMassay. Similarly, any predicted peptide sequence containing an aminoacid residue at a site that is known to be potentially modified in FASNprotein also can be used to assay the extent of modification of FASNprotein in a sample.

FASN fragment peptides may be generated in a variety of ways, includingusing the Liquid Tissue™ protocol described, for example, in U.S. Pat.No. 7,473,532. Liquid Tissue™ protocol and reagents produce peptidesamples suitable for mass spectroscopic analysis from formalin fixedparaffin embedded tissue by proteolytic digestion of the proteins in thetissue/biological sample. Suitable reagents and protocols also arecommercially available from OncoPlexDx (formerly Expression PathologyInc., Rockville, Md.).

In the Liquid Tissue™ protocol the tissue/biological sample is heated ina buffer for an extended period of time (e.g., from about 80° C. toabout 100° C. for a period of time from about 10 minutes to about 4hours) to reverse or release protein cross-linking. The buffer employedis a neutral buffer, (e.g., a Tris-based buffer, or a buffer containinga detergent). Following heat treatment the tissue/biological sample istreated with one or more proteases including, but not limited to,trypsin, chymotrypsin, pepsin, and endoproteinase Lys-C for a timesufficient to disrupt the tissue and cellular structure of saidbiological sample and to liquefy the sample. Exemplary conditions forthe protease treatment are from 30 minutes to 24 hours at a temperaturefrom 37° C. to 65° C.). Advantageously, endoproteases, and particularlycombinations of two or three endoproteases, used either simultaneouslyor sequentially, are employed to liquefy the sample. For example,suitable combinations of proteases can include, but are not limited to,combinations of trypsin, endoproteinase Lys-C and chemotrypsin, such astrypsin and endoproteinase Lys-C. The result of the heating andproteolysis is a liquid, soluble, dilutable biomolecule lysate.Advantageously, this liquid lysate is free of solid or particulatematter that can be separated from the lysate by centrifugation.

Surprisingly, it was found that many potential peptide sequences fromthe FASN protein are unsuitable or ineffective for use in massspectrometry-based SRM/MRM assays for reasons that are not immediatelyevident. As it was not possible to predict the most suitable peptidesfor MRM/SRM assay, it was necessary to experimentally identify modifiedand unmodified peptides in actual Liquid Tissue™ lysates to develop areliable and accurate SRM/MRM assay for the FASN protein. While notwishing to be bound by any theory, it is believed that some peptidesmight, for example, be difficult to detect by mass spectrometry becausethey do not ionize well or produce fragments that are not distinct fromthose generated from other proteins. Peptides may also fail to resolvewell in separation (e.g., liquid chromatography), or may adhere to glassor plastic ware, which leads to erroneous results in the SRM/MRM assay.

FASN peptides found in various embodiments of this disclosure (e.g.,Tables 1 and 2 below) were derived from the FASN protein by proteasedigestion of all the proteins within a complex Liquid Tissue™ lysateprepared from cells procured from formalin fixed cancer tissue. Unlessnoted otherwise, in each instance the protease was trypsin. The LiquidTissue™ lysate was then analyzed by mass spectrometry to determine thosepeptides derived from the FASN protein that are detected and analyzed bymass spectrometry. Identification of a specific preferred subset ofpeptides for mass-spectrometric analysis is based on; 1) experimentaldetermination of which peptide or peptides from a protein ionize in massspectrometry analyses of Liquid Tissue™ lysates, and 2) the ability ofthe peptide to survive the protocol and experimental conditions used inpreparing a Liquid Tissue™ lysate. This latter property extends not onlyto the amino acid sequence of the peptide but also to the ability of amodified amino acid residue within a peptide to survive in modified formduring the sample preparation.

Protein lysates from cells procured directly from formalin(formaldehyde) fixed tissue were prepared using the Liquid Tissue™reagents and protocol. This entails collecting cells into a sample tubevia tissue microdissection followed by heating the cells in the LiquidTissue™ buffer for an extended period of time. Once the formalin-inducedcross linking has been negatively affected, the tissue/cells are thendigested to completion in a predictable manner using a protease such as,trypsin. The skilled artisan will recognize that other proteases, and inparticular, endoproteases may be used in place of, or in addition to,trypsin. Each protein lysate was used to prepare a collection ofpeptides by digestion of intact polypeptides with the protease orprotease combination. Each Liquid Tissue™ lysate was analyzed (e.g., byion trap mass spectrometry) to perform multiple global proteomic surveysof the peptides where the data was presented as identification of asmany peptides as could be identified by mass spectrometry from allcellular proteins present in each protein lysate. An ion trap massspectrometer or another form of a mass spectrometer that is capable ofperforming global profiling for identification of as many peptides aspossible from a single complex protein/peptide lysate may be employed.Ion trap mass spectrometers may, however, be the best type of massspectrometer for conducting global profiling of peptides. AlthoughSRM/MRM assays can be developed and performed on any type of massspectrometer, including a MALDI, ion trap, or triple quadrupole, aninstrument platform for SRM/MRM assay is often considered to be a triplequadrupole instrument platform.

Once as many peptides as possible were identified in a single massspectrometric analysis of a single lysate under the conditions employed,then the list of identified peptides was collated and used to determinethe proteins that were detected in that lysate. This process wasrepeated for multiple Liquid Tissue™ lysates, and the very large list ofpeptides was collated into a single dataset. The resulting datasetrepresents the peptides that can be detected in the type of biologicalsample that was analyzed (after protease digestion), and specifically ina Liquid Tissue™ lysate of the biological sample, and thus includes thepeptides for specific proteins, such as for example the FASN protein.

In one embodiment, the FASN tryptic peptides identified as useful in thedetermination of absolute or relative amounts of the FASN receptorinclude one or more, two or more, three or more, four or more, five ormore, six or more, eight or more, or ten or more of the peptides of SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ IDNO:11, each of which sequences are shown in Table 1. Each of thosepeptides was detected by mass spectrometry in Liquid Tissue™ lysatesprepared from formalin fixed, paraffin embedded tissue. Thus, each ofthe peptides in Table 1, or any combination of those peptides (e.g., oneor more, two or more, three or more, four or more, five or more, six ormore, eight or more, or ten or more of those peptides recited in Table1, and particularly combinations with the peptides found in Table 2) arecandidates for use in quantitative SRM/MRM assay for the FASN protein inhuman biological samples, including directly in formalin fixed patienttissue. Table 2 shows additional information regarding three of thepeptides shown in Table 1.

TABLE 1 SEQ ID Peptide sequence SEQ ID NO: 1 LPEDPLLSGLLDSPALKSEQ ID NO: 2 VGDPQELNGITR SEQ ID NO: 3 DLVEAVAHILGIR SEQ ID NO: 4LQVVDQPLPVR SEQ ID NO: 5 GVDLVLNSLAEEK SEQ ID NO: 6 VLEALLPLKSEQ ID NO: 7 FDASFFGVHPK SEQ ID NO: 8 HGLYLPTR SEQ ID NO: 9 SEGVVAVLLTKSEQ ID NO: 10 VYQWDDPDPR SEQ ID NO: 11 AQVADVVVSR

TABLE 2 Mono Precursor Peptide Isotopic Change Precursor Transition IonSEQ ID sequence Mass State m/z m/z Type SEQ ID NO: 4 LQVVDQPLPVR1262.735 2 632.375 581.376 y5 2 632.375 709.435 y6 2 632.375 824.462 y72 632.375 923.53 y8 2 632.375 1022.599 y9 SEQ ID NO: 6 VLEALLPLK 994.6432 498.329 583.417 y5 2 498.329 654.454 y6 2 498.329 783.497 y7 2 498.329896.581 y8 2 498.329 995.649 y9 SEQ ID NO 9 SEGVVAVLLTK 1114.66 2558.337 573.397 y5 2 558.337 644.434 y6 2 558.337 743.502 y7 2 558.337842.57 y8 2 558.337 899.592 y9 2 558.337 1028.635 y10

The FASN tryptic peptides listed in Table 1 include those detected frommultiple Liquid Tissue™ lysates of multiple different formalin fixedtissues of different human organs including prostate, colon, and breast.Each of those peptides is useful for quantitative SRM/MRM assay of theFASN protein in formalin fixed tissue. Further data analysis of theseexperiments indicated no preference is observed for any specificpeptides from any specific organ site. Thus, each of these peptides issuitable for conducting SRM/MRM assays of the FASN protein on a LiquidTissue™ lysate from any formalin fixed tissue originating from anybiological sample or from any organ site in the body.

In one embodiment, the peptides in Table 1, or any combination of thosepeptides (e.g., one or more, two or more, three or more, four or more,five or more, six or more, eight or more, or ten or more of thosepeptides recited in Table 1, and particularly combinations with thepeptides also found in Table 2) are assayed by methods that do not relyupon mass spectroscopy, including, but not limited to, immunologicalmethods (e.g., Western blotting or ELISA). Regardless of how informationdirected to the amount of the peptide(s) (absolute or relative) isobtained, the information may be employed in any of the methodsdescribed herein, including indicating (diagnosing) the presence ofcancer in a subject, determining the stage/grade/status of the cancer,providing a prognosis, or determining the therapeutics or treatmentregimen for a subject/patient.

Embodiments of the present disclosure include compositions comprisingone or more, two or more, three or more, four or more, five or more, sixor more, eight or more, or ten or more of the peptides in Table 1. Insome embodiments, the compositions comprise the peptides in Table 2.Compositions comprising peptides may include one or more, two or more,three or more, four or more, five or more, six or more, eight or more,or ten or more peptides that are isotopically labeled. Each of thepeptides may be labeled with one or more isotopes selected independentlyfrom the group consisting of: ¹⁸O, ¹⁷O, ³⁴S, ¹⁵N, ¹³C, ²H orcombinations thereof. Compositions comprising peptides from the FASNprotein, whether isotope labeled or not, need not contain all of thepeptides from that protein (e.g., a complete set of tryptic peptides).In some embodiments the compositions do not contain one or more, two ormore, three or more, four or more, five or more, six or more, eight ormore, or ten or more peptides from FASN, and particularly peptidesappearing in Table 1 or Table 2. Compositions comprising peptides may bein the form of dried or lyophized materials, liquid (e.g., aqueous)solutions or suspensions, arrays, or blots.

One consideration for conducting an SRM/MRM assay is the type ofinstrument that may be employed in the analysis of the peptides.Although SRM/MRM assays can be developed and performed on any type ofmass spectrometer, including a MALDI, ion trap, or triple quadrupole,the most advantageous instrument platform for SRM/MRM assay is oftenconsidered to be a triple quadrupole instrument platform. That type of amass spectrometer may be considered to be the most suitable instrumentfor analyzing a single isolated target peptide within a very complexprotein lysate that may consist of hundreds of thousands to millions ofindividual peptides from all the proteins contained within a cell.

In order to most efficiently implement SRM/MRM assay for each peptidederived from the FASN protein it is desirable to utilize information inaddition to the peptide sequence in the analysis. That additionalinformation may be used in directing and instructing the massspectrometer (e.g. a triple quadrupole mass spectrometer) to perform thecorrect and focused analysis of specific targeted peptide(s) such thatthe assay may be effectively performed.

The additional information about target peptides in general, and aboutspecific FASN peptides, may include one or more of the mono isotopicmass of the peptide, its precursor charge state, the precursor m/zvalue, the m/z transition ions, and the ion type of each transition ion.Additional peptide information that may be used to develop an SRM/MRMassay for the FASN protein is shown by example for three (3) of the FASNpeptides from the list in Table 1 and is shown in Table 2. Similaradditional information described for these three (3) FASN peptides shownby example in Table 2 may be prepared, obtained, and applied to theanalysis of the other peptides contained in Table 1.

The method described below was used to: 1) identify candidate peptidesfrom the FASN protein that can be used for a mass spectrometry-basedSRM/MRM assay for the FASN protein, 2) develop individual SRM/MRM assay,or assays, for target peptides from the FASN protein, and 3) applyquantitative assays to cancer diagnosis and/or choice of optimaltherapy.

Assay Method

-   1. Identification of SRM/MRM candidate fragment peptides for the    FASN protein    -   a. Prepare a Liquid Tissue™ protein lysate from a formalin fixed        biological sample using a protease or proteases, (that may or        may not include trypsin), to digest proteins    -   b. Analyze all protein fragments in the Liquid Tissue™ lysate on        an ion trap tandem mass spectrometer and identify all fragment        peptides from the FASN protein, where individual fragment        peptides do not contain any peptide modifications such as        phosphorylations or glycosylations    -   c. Analyze all protein fragments in the Liquid Tissue™ lysate on        an ion trap tandem mass spectrometer and identify all fragment        peptides from the FASN protein that carry peptide modifications        such as for example phosphorylated or glycosylated residues    -   d. All peptides generated by a specific digestion method from        the entire, full length FASN protein potentially can be        measured, but preferred peptides used for development of the        SRM/MRM assay are those that are identified by mass spectrometry        directly in a complex Liquid Tissue™ protein lysate prepared        from a formalin fixed biological sample    -   e. Peptides that are specifically modified (phosphorylated,        glycosylated, etc.) in patient tissue and which ionize, and thus        detected, in a mass spectrometer when analyzing a Liquid Tissue™        lysate from a formalin fixed biological sample are identified as        candidate peptides for assaying peptide modifications of the        FASN protein-   2. Mass Spectrometry Assay for Fragment Peptides from FASN Protein    -   a. SRM/MRM assay on a triple quadrupole mass spectrometer for        individual fragment peptides identified in a Liquid Tissue™        lysate is applied to peptides from the FASN protein        -   i. Determine optimal retention time for a fragment peptide            for optimal chromatography conditions including but not            limited to gel electrophoresis, liquid chromatography,            capillary electrophoresis, nano-reversed phase liquid            chromatography, high performance liquid chromatography, or            reverse phase high performance liquid chromatography        -   ii. Determine the mono isotopic mass of the peptide, the            precursor charge state for each peptide, the precursor m/z            value for each peptide, the m/z transition ions for each            peptide, and the ion type of each transition ion for each            fragment peptide in order to develop an SRM/MRM assay for            each peptide.        -   iii. SRM/MRM assay can then be conducted using the            information from (i) and (ii) on a triple quadrupole mass            spectrometer where each peptide has a characteristic and            unique SRM/MRM signature peak that precisely defines the            unique SRM/MRM assay as performed on a triple quadrupole            mass spectrometer    -   b. Perform SRM/MRM analysis so that the amount of the fragment        peptide of the FASN protein that is detected, as a function of        the unique SRM/MRM signature peak area from an SRM/MRM mass        spectrometry analysis, can indicate both the relative and        absolute amount of the protein in a particular protein lysate.        -   i. Relative quantitation may be achieved by:            -   1. Determining increased or decreased presence of the                FASN protein by comparing the SRM/MRM signature peak                area from a given FASN peptide detected in a Liquid                Tissue™ lysate from one formalin fixed biological sample                to the same SRM/MRM signature peak area of the same FASN                fragment peptide in at least a second, third, fourth or                more Liquid Tissue™ lysates from least a second, third,                fourth or more formalin fixed biological samples            -   2. Determining increased or decreased presence of the                FASN protein by comparing the SRM/MRM signature peak                area from a given FASN peptide detected in a Liquid                Tissue™ lysate from one formalin fixed biological sample                to SRM/MRM signature peak areas developed from fragment                peptides from other proteins, in other samples derived                from different and separate biological sources, where                the SRM/MRM signature peak area comparison between the 2                samples for a peptide fragment are normalized to amount                of protein analyzed in each sample.            -   3. Determining increased or decreased presence of the                FASN protein by comparing the SRM/MRM signature peak                area for a given FASN peptide to the SRM/MRM signature                peak areas from other fragment peptides derived from                different proteins within the same Liquid Tissue™ lysate                from the formalin fixed biological sample in order to                normalize changing levels of FASN protein to levels of                other proteins that do not change their levels of                expression under various cellular conditions.            -   4. These assays can be applied to both unmodified                fragment peptides and for modified fragment peptides of                the FASN protein, where the modifications include but                are not limited to phosphorylation and/or glycosylation,                and where the relative levels of modified peptides are                determined in the same manner as determining relative                amounts of unmodified peptides.        -   ii. Absolute quantitation of a given peptide may be achieved            by comparing the SRM/MRM signature peak area for a given            fragment peptide from the FASN protein in an individual            biological sample to the SRM/MRM signature peak area of an            internal fragment peptide standard spiked into the protein            lysate from the biological sample            -   1. The internal standard is a labeled synthetic version                of the fragment peptide from the FASN protein that is                being interrogated. This standard is spiked into a                sample in known amounts, and the SRM/MRM signature peak                area can be determined for both the internal fragment                peptide standard and the native fragment peptide in the                biological sample separately, followed by comparison of                both peak areas            -   2. This can be applied to unmodified fragment peptides                and modified fragment peptides, where the modifications                include but are not limited to phosphorylation and/or                glycosylation, and where the absolute levels of modified                peptides can be determined in the same manner as                determining absolute levels of unmodified peptides.-   3. Apply Fragment Peptide Quantitation to Cancer Diagnosis and    Treatment    -   a. Perform relative and/or absolute quantitation of fragment        peptide levels of the FASN protein and demonstrate that the        previously-determined association, as well understood in the        field of cancer, of FASN protein expression to the        stage/grade/status of cancer in patient tumor tissue is        confirmed    -   b. Perform relative and/or absolute quantitation of fragment        peptide levels of the FASN protein and demonstrate correlation        with clinical outcomes from different treatment strategies,        wherein this correlation has already been demonstrated in the        field or can be demonstrated in the future through correlation        studies across cohorts of patients and tissue from those        patients. Once either previously established correlations or        correlations derived in the future are confirmed by this assay        then the assay method can be used to determine optimal treatment        strategy

Assessment of FASN protein levels in tissues based on analysis offormalin fixed patient-derived tissue can provide diagnostic,prognostic, and therapeutically-relevant information about eachparticular patient. In one embodiment, this disclosure describes amethod for measuring the level of the FASN protein in a biologicalsample, comprising detecting and/or quantifying the amount of one ormore modified or unmodified FASN fragment peptides in a protein digestprepared from the biological sample using mass spectrometry; andcalculating the level of modified or unmodified FASN protein in thesample; and where the level is a relative level or an absolute level. Ina related embodiment, quantifying one or more FASN fragment peptidescomprises determining the amount of each of the FASN fragment peptidesin a biological sample by comparison to a known amount of an addedinternal standard peptide, where each of the FASN fragment peptides inthe biological sample is compared to an internal standard peptide havingthe same amino acid sequence. In some embodiments the internal standardis an isotopically labeled internal standard peptide comprises one ormore heavy stable isotopes selected from ¹⁸O, ¹⁷O, ³⁴S, ¹⁵N, ¹³C, ²H orcombinations thereof.

The method for measuring the level of the FASN protein in a biologicalsample described herein (or fragment peptides as surrogates thereof) maybe used as a diagnostic indicator of cancer in a patient or subject. Inone embodiment, the results from measurements of the level of the FASNprotein may be employed to determine the diagnostic stage/grade/statusof a cancer by correlating (e.g., comparing) the level of FASN proteinfound in a tissue with the level of that protein found in normal and/orcancerous or precancerous tissues.

Embodiments:

-   1. A method for measuring the level of the Fatty Acid Synthase    (FASN) protein in a biological sample, comprising detecting and/or    quantifying the amount of one or more modified or unmodified FASN    fragment peptides in a protein digest prepared from said biological    sample using mass spectrometry; and calculating the level of    modified or unmodified FASN protein in said sample; and    -   wherein said amount is a relative amount or an absolute amount.-   2. The method of embodiment 1, further comprising the step of    fractionating said protein digest prior to detecting and/or    quantifying the amount of one or more modified or unmodified FASN    fragment peptides.-   3. The method of embodiment 2, wherein said fractionating step is    selected from the group consisting of gel electrophoresis, liquid    chromatography, capillary electrophoresis, nano-reversed phase    liquid chromatography, high performance liquid chromatography, or    reverse phase high performance liquid chromatography.-   4. The method of any of embodiments 1-3, wherein said protein digest    of said biological sample is prepared by the Liquid Tissue™    protocol.-   5. The method of any of embodiments 1-3, wherein said protein digest    comprises a protease digest.-   6. The method of embodiment 5, wherein said protein digest comprises    a trypsin digest.-   7. The method of any of embodiments 1-6, wherein said mass    spectrometry comprises tandem mass spectrometry, ion trap mass    spectrometry, triple quadrupole mass spectrometry, MALDI-TOF mass    spectrometry, MALDI mass spectrometry, and/or time of flight mass    spectrometry.-   8. The method of embodiment 7, wherein the mode of mass spectrometry    used is Selected Reaction Monitoring (SRM), Multiple Reaction    Monitoring (MRM), and/or multiple Selected Reaction Monitoring    (mSRM), or any combination thereof.-   9. The method of any of embodiments 1 to 8, wherein the one or more    modified or unmodified FASN fragment peptides comprise different    amino acid sequences independently selected from those set forth in    SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ    ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and    SEQ ID NO:11.-   10. The method of any of embodiments 1-9, wherein the biological    sample is a blood sample, a urine sample, a serum sample, an ascites    sample, a sputum sample, lymphatic fluid, a saliva sample, a cell,    or a solid tissue.-   11. The method of embodiment 10, wherein the tissue is formalin    fixed tissue.-   12. The method of embodiment 10 or 11, wherein the tissue is    paraffin embedded tissue.-   13. The method of embodiment 10, wherein the tissue is obtained from    a tumor.-   14. The method of embodiment 13, wherein the tumor is a primary    tumor.-   15. The method of embodiment 13, wherein the tumor is a secondary    tumor.-   16. The method of any of embodiments 1 to 15, further comprising    quantifying a modified or unmodified FASN fragment peptide.-   17. The method of embodiment 16, wherein quantifying the FASN    fragment peptide comprises comparing an amount of one or more, two    or more, three or more, four or more, or five or more FASN fragment    peptides comprising an amino acid sequence of about 8 to about 45    amino acid residues of FASN as shown in SEQ ID NO:1, SEQ ID NO:2,    SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ    ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11 in one    biological sample to the amount of the same FASN fragment peptide in    a different and separate biological sample.-   18. The method of embodiment 17, wherein quantifying one or more    FASN fragment peptides comprises determining the amount of the each    of the FASN fragment peptides in a biological sample by comparison    to an added internal standard peptide of known amount, wherein each    of the FASN fragment peptides in the biological sample is compared    to an internal standard peptide having the same amino acid sequence.-   19. The method of embodiment 18, wherein the internal standard    peptide is an isotopically labeled peptide.-   20. The method of embodiment 19, wherein the isotopically labeled    internal standard peptide comprises one or more heavy stable    isotopes selected from ¹⁸O, ¹⁷O, ³⁴S, ¹⁵N, ¹³C, ²H or combinations    thereof.-   21. The method of any of embodiments 1 to 20, wherein detecting    and/or quantifying the amount of one or more modified or unmodified    FASN fragment peptides in the protein digest indicates the presence    of modified or unmodified FASN protein and an association with    cancer in the subject.-   22. The method of embodiment 21, further comprising correlating the    results of said detecting and/or quantifying the amount of one or    more modified or unmodified FASN fragment peptides, or the amount of    said FASN protein to the diagnostic stage/grade/status of the    cancer.-   23. The method of embodiment 22, wherein correlating the results of    said detecting and/or quantifying the amount of one or more modified    or unmodified FASN fragment peptides, or the amount of said FASN    protein to the diagnostic stage/grade/status of the cancer is    combined with detecting and/or quantifying the amount of other    proteins or peptides from other proteins in a multiplex format to    provide additional information about the diagnostic    stage/grade/status of the cancer.-   24. The method of any one of embodiments 1-23, further comprising    selecting for the subject from which said biological sample was    obtained a treatment based on the presence, absence, or amount of    one or more FASN fragment peptides or the amount of FASN protein.-   25. The method of any one of embodiments 1-24, further comprising    administering to the patient from which said biological sample was    obtained a therapeutically effective amount of a therapeutic agent,    wherein the therapeutic agent and/or amount of the therapeutic agent    administered is based upon amount of one or more modified or    unmodified FASN fragment peptides or the amount of FASN protein.-   26. The method of embodiments 24 and 25, wherein the treatment or    the therapeutic agent is directed to cancer cells expressing the    FASN protein.-   27. The method of embodiments 1 to 26, wherein the biological sample    is formalin fixed tumor tissue that has been processed for    quantifying the amount of one or more modified or unmodified FASN    fragment peptides employing the Liquid Tissue™ protocol and    reagents.-   28. The method of any of embodiments 1-27, wherein said one or more    modified or unmodified FASN fragment peptides is two or more, three    or more, four or more, five or more, six or more, eight or more, or    ten or more of the peptides in Table 1.-   29. The method of any of embodiments 1-28, comprising quantifying    the amount of one, two or more, three or more, four or more, five or    more, six or more, eight or more, or ten or more of the peptides in    Table 2.-   30. A composition comprising one, two or more, three or more, four    or more, five or more, six or more, eight or more, or ten or more of    the peptides in Table 1 or antibodies thereto, said composition    optionally excluding one, two, three or more other peptides of FASN.-   31. The composition of embodiment 30, comprising one, two or three    of the peptides of Table 2 or antibodies thereto.

It is to be understood that the description, specific examples,embodiments, and data, while indicating exemplary aspects, are given byway of illustration and are not intended to limit the presentdisclosure. Various changes and modifications within the presentdisclosure will become apparent to the skilled artisan from thediscussion, detailed description and data contained herein, and thus areconsidered part of the subject matter of this application.

The invention claimed is:
 1. A method for measuring the level of theFatty Acid Synthase (FASN) protein in a human biological sample offormalin-fixed tissue, comprising detecting and/or quantifying theamount of an FASN fragment peptide in a protein digest prepared fromsaid human biological sample using mass spectrometry; wherein the FASNfragment peptide is SEQ ID NO:4, and calculating the level of FASNprotein in said sample; and wherein said amount is a relative amount oran absolute amount.
 2. The method of claim 1, further comprising thestep of fractionating said protein digest prior to detecting and/orquantifying the amount of said FASN fragment peptide.
 3. The method ofclaim 2, wherein said fractionating step is selected from the groupconsisting of gel electrophoresis, liquid chromatography, capillaryelectrophoresis, nano-reversed phase liquid chromatography, highperformance liquid chromatography, or reverse phase high performanceliquid chromatography.
 4. The method of claim 1, wherein said proteindigest comprises a trypsin digest.
 5. The method of claim 1, wherein thetissue is paraffin embedded tissue.
 6. The method of claim 1, whereinthe tissue is obtained from a tumor.
 7. The method of claim 1,comprising quantifying said FASN fragment peptide.
 8. The method ofclaim 7, wherein quantifying the FASN fragment peptide comprisescomparing an amount of said FASN fragment peptide in one biologicalsample to the amount of the same FASN fragment peptide in a differentand separate biological sample.
 9. The method of claim 8, whereinquantifying said FASN fragment peptide comprises determining the amountof said FASN fragment peptide in a biological sample by comparison to anadded internal standard peptide of known amount, wherein said FASNfragment peptide in the biological sample is compared to an internalstandard peptide having the same amino acid sequence, and wherein theinternal standard peptide is an isotopically labeled peptide.